The developing tooth is a valuable paradigm for understanding the genetic and molecular basis for the way in which morphogenesis and terminal differentiation are achieved during organogenesis. Tooth morphogenesis involves reciprocal signaling interactions between the dental epithelium and mesenchyme mediated by transcription factors and growth factors, as well as cell surface and extracellular matrix (ECM) molecules. As mitogenic factors, fibroblast growth factors (FGFs) play essential roles in coupling dental epithelial and mesenchymal growth, since epithelial FGFs mainly regulate dental mesenchymal growth and mesenchymal FGFs regulate epithelial growth. Twist1 and Twist2 belong to the evolutionarily conserved Twist family of basic helix-loop-helix (bHLH) transcription factors and are highly expressed in the dental mesenchyme during tooth morphogenesis. Their expression in the dental mesenchyme can be induced by epithelial FGFs, and studies in our laboratory have shown that Twist1, together with its heterodimeric binding partner, E12, regulates FGF receptor 2 (Fgfr2) and Fgf10 promoter activities. These observations suggest that epithelial FGFs and mesenchymal FGFs might be coupled through Twist1 and Twist2 in the dental mesenchyme. The long-term goal of this research project is to understand the molecular mechanisms governing tooth morphogenesis and terminal differentiation of odontoblasts. The proposed studies will test the hypothesis that Twist1 forms heterodimers with E12 in the dental mesenchyme, and modulates tooth morphogenesis by regulating the expression of Fgfr2 and Fgf10. To test this hypothesis, two specific aims are proposed. Aim 1 will determine whether Twist1, along with its heterodimeric binding partner E12, regulates the expression of Fgfr2 and Fgf10 at the transcriptional level using in vitro approaches including quantitative real-time PCR, chromatin immunoprecipitation (ChIP) assay, and deletion analyses of the Fgfr2 and Fgf10 promoters. Aim 2 will determine if alterations in the expression of Twist1 and Twist2 affect tooth morphogenesis and odontoblast differentiation in vivo. This aim will be achieved by generating and analyzing the Twist1 and Twist2 compound mutant embryos to examine whether there are Twist dosage-dependent changes in the expression of Fgfr2 and Fgf10 in the dental mesenchyme as well as phenotypic alterations in the mutant teeth. Successful completion of the proposed studies will show that Twist1 and Twist2 play key roles in mediating FGF signaling triggered by epithelial FGFs and in generating recursive mesenchymal FGFs in the dental mesenchyme during advancing tooth morphogenesis (bud to early bell stages). PUBLIC HEALTH RELEVANCE: Successful completion of the proposed studies will provide fundamental information on the molecular mechanisms that regulate tooth morphogenesis and will contribute new insights about how teeth are formed. This information is essential for understanding the pathogeneses of congenital tooth defects, establishing scientifically based treatment modalities, and for developing methods leading to tooth regeneration.